Transcriptomic Regulatory Mechanisms of Isoflavone Biosynthesis in Trifolium pratense

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General Structural Review

Strengths:

The manuscript presents a standard and logical structure (Introduction, Materials and Methods, Results, Discussion).

The narrative is clear and follows a coherent thread: from the biological problem to the identification of key regulatory genes.

Aspects to improve:

Some results sections tend to be overly descriptive without sufficient immediate interpretation; suggestion: integrate brief preliminary interpretations in results subsections.

In some parts, the paragraphs are extensive and could be segmented to improve readability.

Introduction

Strengths:

Provides solid context on the importance of isoflavonoids and their agricultural, nutritional, and pharmacological relevance.

Presents detailed background on key enzymes and involved metabolic pathways.

Suggestions:

Although relevant works on soybean and alfalfa are cited, there is a lack of more explicit discussion on the current state of knowledge in T. pratense. What previous studies exist specifically on this species?

Critical question: Why was red clover (T. pratense) chosen and not another legume with a more consolidated genomics background such as Medicago truncatula?

Materials and Methods

Strengths:

Detailed description of plant growth, greenhouse conditions, experimental design, and RNA and isoflavone extraction protocols.

Appropriate analytical tools are used: RNA-seq, WGCNA, DESeq2, qRT-PCR.

Critical questions and recommendations:

How many biological replicates were used in RNA-seq and isoflavone quantification? Although three are mentioned, it would be helpful to reiterate this in the corresponding subsection. Include in the manuscript.

A K-means clustering analysis was applied to group 500 accessions. What was the K value used and how was it justified? Include in the manuscript.

Validation with qRT-PCR included 15 genes, but how were these genes selected? Based on their centrality in WGCNA networks, or on their logFC? Include in the manuscript.

The use of HISAT2 is mentioned, but the mapping percentage and average genome/transcriptome coverage are not detailed. This is important to assess the robustness of the analysis. Include in the manuscript.

Results

Strengths:

Excellent integration of multi-stage analysis (isoflavone content  transcriptomics, co-expression , validation).

Clear presentation of transcriptional differences between H and L groups across developmental stages.

Technical critiques and suggestions:

The high number of DEGs (>10,000 in L1 vs H1) could reflect biological noise or biases in the analysis. Was batch effect controlled? Describe and justify in the manuscript.

Discuss more thoroughly the decrease in the number of DEGs at the flowering stage. Is it due to transcriptional stabilization or a shift in metabolic priority? Discuss and justify.

The interpretation of WGCNA modules is solid, but figures or descriptions of hub genes are lacking. Which central genes have higher connectivity within the MEturquoise and MEyellow modules? Discuss and justify.

The importance of PAL, CHS, IFS, etc. is mentioned, but quantitative values (logFC, FPKM, or TPM) are not shown. It would be useful to include a summary table with these values for the key genes.

Discussion

Strengths:

The activation of specific metabolic pathways in high isoflavone content groups is appropriately interpreted.

The role of transcription factors such as MYB, WRKY, and NAC is well contextualized.

Aspects to strengthen:

Add a direct discussion/comparison with previous studies in other species (soybean, alfalfa). Are the co-expression modules conserved?

The study’s limitations are not addressed: for example, the omission of proteomic or metabolomic levels, or the possible influence of epigenetic variability.

Open question: how could this transcriptomic information be integrated into T. pratense breeding programs? Are there available gene editing tools or SNP markers linked to these genes?

Comments on the Quality of English Language

Improve English writing and connectors

Author Response

We sincerely thank the reviewers for their thoughtful and constructive comments. We greatly appreciate your positive comments on the structure, clarity, and methodological rigor of the manuscript. Based on your suggestions, we have revised the manuscript to improve readability and depth of interpretation. We have added brief explanations after the key results to enhance the biological relevance of the findings and divided overly long paragraphs to improve narrative clarity. The Introduction has been expanded to include the specific study of Trifolium pratense (clover) and to clarify the rationale for choosing this species over model legumes such as alfalfa, emphasizing its naturally rich isoflavone content and its importance as a forage crop. In the Materials and Methods section, we explicitly state that three biological replicates were used for RNA-seq and isoflavone quantification, and we have added the rationale for the choice of K value in K-means clustering. In addition, we clarified the gene selection criteria for qRT-PCR validation. We thank the reviewers for their insightful suggestions on the magnitude of DEGs, control of batch effects, interpretation of DEG dynamics across developmental stages, and inclusion of hub gene analysis and key gene expression tables. These are all good points and we plan to discuss them in detail in the next round of revisions. We thank the reviewers for their comments, which guided us to further improve the manuscript in terms of scientific rigor and clarity.

Reviewer 2 Report

Comments and Suggestions for Authors

This work is highly novel and very interesting. English could be improved.

1. This work is about the detection of isoflavone content in three groups of the herb Trifolium pratense showing higher levels of isoflavones compared with three other groups containing lower amounts of these compounds. The groups showing the highest and the lowest amounts of isoflavones were chosen to perform transcriptomic analyses. The two selected groups in vegetative stage, flowering stage and seed productive stage of T. pretense were analyzed through RNAseq to quantify the expression of enzymes of the phenylpropanoid pathway including those involved in isoflavone synthesis. The expression of enzymes PAL, C4H, 4CL, CHS and IFS were increased in the high-productive T. pratense as well as a MAPK, transcription factors (TF) MYB, WRKY and NAC, enzyme involved in phytohormone synthesis, sugar metabolim and circadian rhythm. The increased expression of several up-regulated genes was verified using qRT-PCR. 

2. Transcriptomic analyses have been performed in other plant species such as soybean and alfalfa regarding phenylpropanoid metabolism and TFs but not in red clover (T. pratense) that is used in animal feeding. 

3. The methodology is well written. Conclusions are in accord with observed experimental results. References are appropiate. No more comments on Tables and figures. 

Some minor modifications and some explanations are required in the text:

Line 120, 1 mg mL-¹

Line 175, the size of the letters of primer niucleotides should be reduced

Line 170, actin

Line 180, (Fig. 1)

Line 192, Why did you choose H6 as the group containing the lowest level of isoflavones when H5 showed the lowest level in Table 2?

Line 227, (Fig. 2A-C)

Line 244-256, indicate at the end of the corresponding sentence (Fig. 1A), (Fig. 1B) annd (Fig. 1C)

Line 267, (Fig. 3) and corect all over the text

Line 421, T. pratense and also in lines 496, 537, 555

Line 506, (Fig. 9) not 99

Line 689, do not repeat Weighted Gene Co-expression Network

Line 690, T. pratense

Line 718, Camellia sinensis in italics

 

Author Response

We sincerely thank the reviewer for the encouraging feedback and thoughtful comments regarding the novelty and significance of our work. We are pleased that the overall design, methodology, and scientific rigor of our study were well received. In accordance with your suggestions, we have carefully revised the manuscript to address the noted issues. The English language throughout the manuscript has been improved to enhance readability and clarity. We corrected the formatting and notation errors, including the concentration unit in Line 120 ("1 mg·mL⁻¹"), primer font size and consistency in Line 175, corrected the gene name "actin" in Line 170, and added appropriate figure citations (e.g., Fig. 1, Fig. 2A–C, Fig. 3, Fig. 9) throughout the Results section for clarity. The scientific name T. pratense has been consistently italicized across the manuscript (Lines 421, 496, 537, 555, 690), and "Camellia sinensis" in Line 718 has also been italicized. Regarding Line 192, we thank the reviewer for catching the inconsistency in isoflavone content reporting; we have double-checked the dataset and clarified the rationale for selecting H6 as the representative low-isoflavone group. Additionally, we removed the redundant repetition of the full term "Weighted Gene Co-expression Network" in Line 689. We sincerely appreciate your constructive feedback, which helped us improve the quality and precision of our manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

This study presents a comprehensive transcriptomic analysis of isoflavone biosynthesis in Trifolium pratense, offering valuable insights into the genetic and molecular mechanisms involved. The identification of key gene modules and transcriptional regulators provides a solid foundation for future work in metabolic engineering and breeding. However, the clarity and presentation of figures and supporting data need further revision to enhance readability and interpretation. Improved visual representation and more detailed legends would help strengthen the manuscript’s impact and accessibility.

Major comments:

1. In lines 100–101, the growth cycle of red clover is categorized into three stages: vegetative growth, flowering, and seed maturation, defined as stage 1, 2, and 3, respectively. The high-isoflavone (H) and low-isoflavone (L) groups are accordingly labeled as H1/L1, H2/L2, and H3/L3. However, this naming convention is not consistent throughout the manuscript. For example, in line 181, H1–H6 are used to describe six distinct groups, which causes confusion as H1, H2, and H3 are already used to represent developmental stages. To improve clarity for the reader, it is recommended to revise the naming of the six distinct groups introduced in line 181 to avoid overlapping terminology.
2. The gene IDs listed in Table 1 are not consistent with those presented in Figure 10. Specifically, genes numbered 4, 12, 13, 14, and 15 in Table 1 are missing from Figure 10, while some gene IDs shown in Figure 10 are not mentioned in Table 1. In line 166, the authors state that 15 genes were randomly selected, yet only 14 appear in Figure 10. This discrepancy should be clarified. Additionally, the term "reference gene" is mentioned, but it is unclear what gene was used as the reference and how gene expression was normalized or compared.

In line 172, it is mentioned that each sample was analyzed in three biological replicates, and statistical significance was assessed using one-way ANOVA (GraphPad Prism 9.0). However, Figure 10 does not display any error bars, which are essential for representing variability across replicates. The authors are encouraged to include error bars in the figure and provide complete information in the figure legend, including the type of error bars used (e.g., standard deviation or standard error) and a description of the statistical analysis.

In line 546, the genes emrTU_0115460 and emrTU_0121170, and in line 548, emrTU_0121175 and emrTU_0131120, are not consistent with the gene names listed in Table 1 and Figure 10. Please ensure that gene names are consistent throughout the manuscript.

The authors are encouraged to provide more specific information regarding the methodology used for expression comparison.

3. In line 96, it is stated that a total of 500 red clover germplasm resources were cultivated. Could the authors clarify how these germplasm resources were obtained? Were they collected from natural populations, sourced from germplasm banks, or acquired through other means? Providing this information would enhance the transparency and reproducibility of the study.
4. In lines 105 and 566, it is stated that the group with the lowest isoflavone content was selected for analysis. However, according to Table 2, the selected group H6 (mentioned in line 198) is not the one with the lowest isoflavone content—H5 appears to have the lowest value. The authors are requested to clarify this inconsistency and ensure the selection criteria are accurately reflected in both the text and the data.
5. Please add the six distinct groups mentioned in line 181 to Figure 1 to enhance clarity and consistency between the text and the figure. Additionally, the figure legend should be expanded to provide a more detailed explanation of the grouping criteria and any relevant context for interpretation.
6. In Figure 2, it appears that sample L2.3 is missing from panel (a). Could the authors clarify why this sample is not included and whether this was intentional or due to a data omission?
7. Typo in line 506: "Figure 99" should be corrected to "Figure 9".

Author Response

1. Inconsistency in the use of H1–H3 and H1–H6

Reviewer comment: Lines 100–101 label developmental stages as H1/L1 to H3/L3, but H1–H6 is later used to describe six different groups, which is confusing.

Author response: We thank the reviewer for pointing out the inconsistency in terminology. To avoid confusion, we have renamed the six germplasm clusters originally labeled as H1–H6 to G1–G6. The terms H1/L1, H2/L2, and H3/L3 are now exclusively used to refer to high- and low-isoflavone groups at the vegetative, flowering, and seed maturation stages, respectively. These designations have been corrected throughout the manuscript, figures, and legends to improve clarity.

 

2. Inconsistency between Table 1 and Figure 10; reference gene not specified

Reviewer comment: Gene IDs in Table 1 do not match those in Figure 10, and only 14 genes are shown in the figure instead of 15 as stated. The reference gene is also not specified.

Author response: We appreciate the reviewer’s important suggestion. We have revised Table 1 to ensure consistency with the gene IDs presented in Figure 10. Additionally, we have clarified that RCD1 (RADICAL-INDUCED CELL DEATH1) was used as the internal reference gene for qRT-PCR analysis, and expression levels were calculated using the 2^(-ΔΔCt) method. This information has been added to the Materials and Methods section.

 

3. Error bars missing in Figure 10

Reviewer comment: Figure 10 lacks error bars, making it difficult to assess biological variation.

Author response: We thank the reviewer for highlighting this issue. We have added standard deviation (SD) error bars to Figure 10, based on three biological replicates.

 

4. Inconsistent gene names between text, Table 1, and Figure 10

Reviewer comment: Gene names in lines 546 and 548 do not match those in Table 1 and Figure 10.

Author response: We appreciate the reviewer’s attention to detail. We have revised the relevant gene IDs in the main text to ensure consistency with Table 1 and Figure 10.

 

5. Expression comparison method not clearly described

Reviewer comment: Please clarify how gene expression comparisons were conducted.

Author response: Thank you for the suggestion. We have added details to the Materials and Methods section, specifying that differential expression analysis was performed using DESeq2 with thresholds of |log₂FC| ≥ 1 and adjusted p < 0.05. For qRT-PCR validation, the 2^(-ΔΔCt) method was used with RCD1 as the normalization gene.

 

6. Unclear description of germplasm sources

Reviewer comment: Please specify the origin of the 500 red clover accessions.

Author response: We thank the reviewer for raising this point. We have added a clarification in the Materials and Methods section stating that the 500 red clover germplasm accessions were mainly collected from natural populations in northern and western China, encompassing a variety of ecological environments. All materials were identified, propagated, and cultivated under uniform greenhouse conditions to ensure experimental consistency.

 

7. Unclear rationale for selecting H6 as the lowest-isoflavone group; Table 2 shows H5 has the lowest content

Reviewer comment: Lines 105 and 566 state that the group with the lowest isoflavone content was selected, but Table 2 shows H5 has a lower content than H6.

Author response: We thank the reviewer for identifying this inconsistency.

 

8. G1–G6 not labeled in Figure 1; legend lacks sufficient explanation

Reviewer comment: Please add G1–G6 labels to Figure 1 and expand the legend.

Author response: In accordance with the reviewer’s suggestion, we have added G1–G6 group labels to Figure 1 and used colors to distinguish different clusters. The legend has also been expanded to explain the grouping criteria (hierarchical clustering based on isoflavone content) and the biological significance of each group.

 

9. Sample L2.3 missing in Figure 2(a)

Reviewer comment: Sample L2.3 appears to be missing in Figure 2(a). Was this intentional or an oversight?

Author response: We thank the reviewer for noting the absence of sample L2.3 in Figure 2(a). This omission was intentional and based on our experimental design. L2.3 exhibited an anomalous expression profile inconsistent with its biological replicates, likely due to RNA degradation or technical issues during extraction. To ensure accuracy and consistency of the analysis, we excluded L2.3 from Figure 2(a).

 

10. Typographical error in line 506

Reviewer comment: Typo: “Figure 99” should be “Figure 9”.

Author response: Thank you for pointing this out. We have corrected the error by changing “Figure 99” to “Figure 9” in line 506.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

All concerns have been adequately addressed by the authors. I have no additional comments.